Abstract Double-strand breaks (DSBs) are one of the most toxic types of DNA damage that occur within the cell. If left unrepaired they can lead to loss of genetic information and overall genome instability. In yeast the major pathway that repairs DSBs is homologous recombination (HR), where the lesion is repaired using a template that shares homology with the original sequence. All mitotic recombination studies, to date, have utilized the site specific enzymes HO or I-SceI to produce DSBs with 3? overhangs. However, DSBs can have various end structures in vivo. In this study we explore the impact different end structures have on intermediates and outcomes of HR. We will compare the traditional 3? overhang created by I-SceI to a 5? overhang created by a Zinc Finger Nuclease (ZFN) and a blunt end created by Cas9. An ectopic chromosomal recombination assay, will be used in which we engineered single nucleotide polymorphisms into the donor allele, allowing us to identify donor and recipient strands present in heteroduplex DNA (hetDNA) repair intermediates. Preliminary data show differences between I-SceI induced DSBs and ZFN induced DSBs with respect to the ratio of noncrossover (NCO) to crossovers (CO) outcomes among recombination products. The work outlined in this proposal will add to our growing knowledge of the HR mechanism beyond the repair of DSBs with traditional 3? overhangs. Also these findings have the potential to inform HR mediated genome editing and eventually gene therapy.